METHOD FOR PRODUCING FERMENTED MILK USING NOVEL LACTIC ACID BACTERIA

Abstract

ABSTRACT The present invention relates to a method for producing a fermented milk, including: performing fermentation using both bacteria belonging to the genus Bifidobacterium and bacteria belonging to the genus Lactococcus as lactic acid bacteria, wherein the bacteria belonging to the genus Lactococcus have the following bacteriological properties: (1) fermentability which curdles a 10% (WAV) reconstituted skim milk medium when cultivated at a temperature of 25°C to 37°C for 16 hours; (2) Bifidobacterium longum growth-promoting properties which lead to a viable count of Bifidobacterium longum of 5×108 CFU/g or more, when co-cultivated with Bifidobacterium longum in the 10% (W/W) reconstituted skim milk medium until the pH thereof is 4.4 to 4.6; and (3) Bifidobacterium longum survivability-improving properties during storage, which lead to a survival rate of Bifidobacterium longum of 30% or more, after co-cultivation with Bifidobacterium longum in the 10% (W/W) reconstituted skim milk medium until the pH thereof is 4.4 to 4.6, rapid cooling, and two weeks storage at 10°C, and also relates to a fermented milk prepared by the production method.

Full Text

TECHNICAL FIELD
[0001]
The present invention relates to a method for producing fermented milk using
novel lactic acid bacteria belonging to the genus Lactococcus, and fermented milk
prepared in accordance with the production method.
Priority is claimed on Japanese Patent Application No. 2007-032646, filed on
February 13, 2007, the contents of which are incorporated herein by reference.
BACKGROUND ART
[0002]
Bacteria belonging to the genus Bifidobacterium (hereinafter, abbreviated to
"Bifidobacterium"), such as Bifidobacterium longum, is one of predominant bacterial
strains in intestinal microflora formed in the human intestinal tract. It is known that
Bifidobacterium have an intestinal function-regulating activity, an immuno-stimulating
activity, and an anti-cancer activity. Accordingly, demands for food products containing
viable Bifidobacterium such as milk fermented with Bifidobacterium or the like are
increasing in accordance with an increase of health consciousness of consumers.
[0003]
Bifidobacterium exhibits a poor proliferation potency in a milk medium.
Accordingly, various growth-stimulating substances are generally formulated in
fermented milk so that the Bifidobacterium is contained therein at a constant content, for
example, 1x107 CFU/ml. However, the growth-stimulating substances are generally
expensive and may degrade the taste. In addition, preservation of Bifidobacterium
under acidic conditions is difficult and tends to result in death of Bifidobacterium. Thus,

the viable count of the Bifidobacterium in fermented milk products decreases with
accelerating speed during the course of distribution of the fermented milk products.
Accordingly, it is expected that a promotion of the growth of Bifidobacterium or
an improvement of the survivability thereof during storage enables not only preparation
of fermented milk containing a large amount of viable Bifidobacterium, but also
preparation of fermented milk which can keep an abundant amount of viable
Bifidobacterium from immediately after preparation until being consumed.
[0004]
Various methods for promoting the growth of Bifidobacterium or improving the
survivability thereof during storage by fermentation with Bifidobacterium and another
lactic acid bacterium without adding any growth-stimulating substances or the like have
been disclosed. For example, (1) yogurt containing Lactococcus lactis subsp. lactis,
Lactococcus lactis subsp. cremoris, and Bifidobacterium, and a method for preparing the
yogurt have been disclosed (see, for example, Patent Document 1), with respect to a
method for promoting the growth of Bifidobacterium to prepare fermented milk.
[0005]
For example, (2) a method for fermenting milk with Bifidobacterium, including
cultivating Bifidobacterium breve together with Lactococcus lactis subsp. lactis, which
forms neither diacetyl nor acetoin, in a medium containing milk as the main component
thereof has been disclosed (see, for example, Patent Document 2), with respect to a
method for improving the survivability of Bifidobacterium during storage of fermented
milk.
Patent Document 1: Japanese Patent Publication No. 3,364,491.
Patent Document 2: Japanese Patent Publication No. 3,068,484.
DISCLOSURE OF THE INVENTION
[Problems to be Solved by the Invention]
[0006]
Although the growth of Bifidobacterium is promoted and the fermentation time

is shortened in accordance with the above-mentioned method (1), there is no disclosure
with respect to the survivability of Bifidobacterium during storage in Patent Document 1.
In contrast, although both growth-stimulating effects and survivability-improving effects
are recognized by using a mixture composed of a specific Bifidobacterium and a specific
lactic acid bacterium in accordance with the above-mentioned method (2), there is no
disclosure with respect to Bifidobacterium other than Bifidobacterium breve, such as
Bifidobacterium longum, which is generally formulated in food products.
[0007]
The present invention has for an object thereof the provision of a method for
producing fermented milk using lactic acid bacteria which stimulate the growth of
Bifidobacterium and improve the survivability thereof during storage, and fermented
milk prepared in accordance with the production method.
[Means for Solving the Problems]
[0008]
The inventors of the present invention intensively investigated so as to solve the
above-mentioned problems, and performed a fermentation test by performing
co-cultivation with Bifidobacterium longum to find out lactic acid bacterial strains which
exhibit excellent fermentability in 10% (W/W) reconstituted skim milk medium. As a
result, the inventors found lactic acid bacterium strains which can promote the growth of
Bifidobacterium longum to a viable count of 5×108 CFU/g when pH is 4.4 to 4.6, and
enhance the survival rate of Bifidobacterium longum to 30% or more when stored at
10°C for two weeks after the fermentation is ended and then immediately rapid cooling is
performed. In addition, the present inventors found that fermented milk containing a
great amount of Bifidobacterium and having an excellent survivability during storage can
be produced by using the lactic acid bacteria, and thus the present invention has been
completed.
[0009]
That is, the present invention provides a method for producing fermented milk

including: performing fermentation using, as lactic acid bacteria, both bacteria belonging
to the genus Bifidobacterium and bacteria belonging to the genus Lactococcus having the
following bacteriological properties:
(1) fermentability which curdles a 10% (W/W) reconstituted skim milk medium
when cultivated at a temperature of 25°C to 37°C for 16 hours;
(2) Bifidobacterium longum growth-promoting properties which lead to a viable
count of Bifidobacterium longum of 5×108 CFU/g or more, when co-cultivated with
Bifidobacterium longum in the 10% (W/W) reconstituted skim milk medium until the pH
thereof is 4.4 to 4.6; and
(3) Bifidobacterium longum survivability-improving properties during storage,
which lead to a survival rate of Bifidobacterium longum of 30% or more, after
co-cultivation with Bifidobacterium longum in the 10% (W/W) reconstituted skim milk
medium until the pH thereof is 4.4 to 4.6, rapid cooling, and two weeks storage at 10°C.
The present invention also provides a method for producing fermented milk,
characterized in that the bacteria belonging to the genus Lactococcus have no ability to
ferment xylose and produce neither diacetyl nor acetoin.
The present invention also provides a method for producing fermented milk,
characterized in that the bacteria belonging to the genus Lactococcus are Lactococcus
lactis subsp. lactis.
The present invention also provides a method for producing fermented milk,
characterized in that the bacteria belonging to the genus Lactococcus include at least one
bacterial strain selected from the group consisting of Lactococcus lactis subsp. lactis
MCC852 (FERM BP-10742), Lactococcus lactis subsp. lactis MCC857 (FERM
BP-10757), Lactococcus lactis subsp. lactis MCC859 (FERM BP-10744), Lactococcus
lactis subsp. lactis MCC865 (FERM BP-10745), and Lactococcus lactis subsp. lactis
MCC866 (FERM BP-10746).
The present invention also provides a method for producing fermented milk,
characterized in that the bacteria belonging to the genus Bifidobacterium are
Bifidobacterium longum.

15707, and the like. Bifidobacterium longum FERM BP-7787 is particularly preferable,
since Bifidobacterium longum FERM BP-7787 exhibits excellent fermentability in a milk
medium, excellent acid-resistance during the course of distribution, and excellent gastric
acid-resistance. Bifidobacterium longum FERM BP-7787 was accepted by the
International Patent Organism Depositary, National Institute of Advanced Industrial
Science and Technology, (Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken,
JAPAN (postal code number: 305-8566)) on October 31, 2001.
[0013]
The bacteria belonging to the genus Lactococcus to be used in the present
invention have the properties (1), (2), and (3).
The property (1) relates to fermentability. If a lactic acid bacteria can rapidly
proliferate and have a strong fermentability sufficient to curdle a 10% (W/W)
reconstituted skim milk medium when cultivated therein at a temperature between 25°C
and 37°C for 16 hours, the lactic acid bacteria can effectively promote the growth of
Bifidobacterium, particularly Bifidobacterium longum, at the time of preparing fermented
milk. As used herein, the phrase "curdle a culture medium" refers to a phenomenon in
which the pH of the culture medium decreases below an isoelectric point of a milk
protein thereof by acid fermentation, and thereby the milk protein agglomerates and the
culture medium is curdled. The "10% (W/W) reconstituted skim milk medium" may be
prepared, for example, by dissolving 10% by mass of skim milk powders (manufactured
by Morinaga Milk Industry Co., Ltd., for example) in water.
[0014]
Although the temperature range suitable for fermentation with bacteria
belonging to the genus Lactococcus is generally between 20°C and 30°C, the bacteria
belonging to the genus Lactococcus to be used in the present invention exhibit a strong
fermentability at a temperature between 25°C and 37°C. In other words, the bacteria
belonging to the genus Lactococcus to be used in the present invention exhibit a
sufficient fermentability within a temperature range suitable for fermentation with
Bifidobacterium, particularly with Bifidobacterium longum (30°C to 40°C).

The present invention also provides a method for producing fermented milk,
characterized in that a bacterial strain of the Bifidobacterium longum is Bifidobacterium
longum FERM BP-7787.
The present invention also provides a method for producing fermented milk,
characterized in that both Streptococcus thermophilus and Lactobacillus bulgaricus are
further used as the lactic acid bacteria.
The present invention also provides fermented milk prepared by any of the
above-mentioned method for producing fermented milk.
[Effects of the Invention]
[0010]
In accordance with the method for producing fermented milk according to the
present invention, fermented milk containing a great amount of Bifidobacterium,
particularly Bifidobacterium longum can be efficiently produced as never before. In
addition, the fermented milk according to the present invention maintains a sufficient
viable count of Bifidobacterium, particularly Bifidobacterium longum, even during the
course of distribution, and therefore the fermented milk exhibit further high intestinal
function-regulating effects and is useful for health control.
BEST MODE FOR CARRYING OUT THE INVENTION
[0011]
The present invention relates to a method for producing fermented milk,
characterized in that milk is fermented with both Bifidobacterium and bacteria belonging
to the genus Lactococcus having the above-mentioned properties (1), (2), and (3). In
particular, the method is suitable for production of fermented milk by fermenting milk
using Bifidobacterium longum.
[0012]
Examples of Bifidobacterium longum available in the present invention include
Bifidobacterium longum FERM BP-7787, Bifidobacterium longum-type strain ATCC

The property (2) relates 'to Bifidobacterium longum growth-promoting properties.
A milk medium such as a 10% (W/W) reconstituted skim milk medium exhibits excellent
taste, mouth-feeling, and external appearance, when the pH thereof reaches
approximately 4.6, casein and other components contained therein are generally
precipitated, and the culture medium is wholly curdled. Accordingly, fermentation is
generally stopped by rapid cooling or the like when the pH reaches approximately 4.6, to
prepare fermented milk products. Therefore, the lactic acid bacteria having the
growth-promoting properties that can lead the viable count of Bifidobacterium longum
being a high count of 5>
longum in the 10% (W/W) reconstituted skim milk medium until pH thereof is 4.4 to 4.6
can effectively increase the viable count of Bifidobacterium, particularly Bifidobacterium
longum, in fermented milk at the time of preparation of fermented milk.
[0016]
The property (3) relates to Bifidobacterium longum survivability-improving
properties during storage. The quality preservation time period of fermented milk
products is generally about two weeks when stored at 10°C or lower. Accordingly,
fermented milk which maintains a sufficient viable count of Bifidobacterium, particularly
Bifidobacterium longum, even at the end of the time period by which the quality
preservation thereof is ensured, can be produced, provided that the lactic acid bacteria
having the survivability-improving properties during storage that can lead to a survival
rate of Bifidobacterium longum being 30% or more after co-cultivation with
Bifidobacterium longum in the 10% (W/W) reconstituted skim milk medium until the pH
thereof is 4.4 to 4.6, rapid cooling, and two weeks storage at 10°C.
[0017]
The bacteria belonging to the genus Lactococcus to be used in the present
invention may be prepared in accordance with the following method, for example. First,
bacterial strains are isolated from various samples, and strains which exhibit excellent
fermentability in the 10%'(W/W) reconstituted skim milk medium, more specifically

fermentability sufficient to curdle the 10% (W/W) reconstituted skim milk medium when
cultivated therein at a temperature of 25 to 37°C for 16 hours, are selected from the
isolated bacterial strains. Then, the selected bacterial strains are co-cultivated with
Bifidobacterium longum, and bacterial strains which have Bifidobacterium longum
growth-promoting properties and Bifidobacterium longum survivability-improving
properties during storage defined as the above-mentioned properties (2) and (3) are
selected. It is further preferable that bacterial strains which do not have an ability to
ferment xylose and produce neither diacetyl nor acetoin be selected.
[0018]
In the following, the present invention will be explained in more detail.
1. Isolation of bacterial strains
In order to isolate bacterial strains having the above-mentioned properties from
the natural world, the present inventors collected samples from the natural world in Japan,
diluted the samples with an anaerobic dilution buffer ("The World of Enterobacteria"
published by Soubunsha Co., Ltd., written by Tomotari Mitsuoka, Page 322, 1980:
hereinafter, abbreviated to "Reference 1"), inoculated the diluted samples on each plate
of Briggs liver broth (see the above-mentioned Reference 1, Page 319), and then cultured
the inoculated samples at 30°C under anaerobic conditions. Among the thus obtained
colonies, bacterial strains which showed morphological characteristics of streptococcal
bacteria and were recognized as Gram-positive bacteria by microscopic observation of
applied specimens were picked up. The picked up strains were each streak-inoculated
on each BL agar flat plate and then repeatedly cultivated under anaerobic conditions in
the same manner as described above to obtain purely isolated bacterial strains. The
isolated bacterial strains were subjected to a fermentation test on a 10% (W/W)
reconstituted skim milk medium as described below to obtain 20 bacterial strains with
fermentability defined as the above-mentioned property (1). Then, the obtained
bacterial strains were co-cultured with Bifidobacterium longum to obtain 5 bacterial
strains having both the growth-promoting properties which raise the viable count of
Bifidobacterium longum at a pH of 4.4 to 4.6 to 5×108 CFU/g or more and the

survivability-improving properties during storage which raise the survival rate of
Bifidobacterium longum to 30% or more when stored at 10°C for two weeks after rapid
cooling at a pH of 4.4 to 4.6. The 5 bacterial strains are named as "MCC852",
"MCC857", "MCC859", "MCC865", and "MCC866", respectively.
[0019]
2. Bacteriological properties
The bacteriological properties of the 5 bacterial strains will be shown in the
following. The tests for determining the bacteriological properties were performed with
reference to Bergey's Manual of Systematic Bacteriology, edited by Peter H. A. Sneath,
Vol. 2, published by Williams and Wilkins Company, 1986).
[0020]
(I) Bacterial morphology (observed through an optical microscope after anaerobic
cultivation on a BL agar flat plate at 30°C for 72 hours)
Size: 1 to 2 µm (diameter)
Morphology: Streptococcal bacteria
(II) Gram staining: Positive
(III) Litmus milk: Curdled
(IV) Endospore formation: Negative
(V) Gas production from glucose: Negative
(VI) Motility: Negative
(VII) Catalase activity: Negative
(VIII) Arginine decarboxylase test: Positive
(IX) Gas production from citric acid: Negative
(X) Temperature susceptibility (at 60°C for 30 minutes and at 65°C for 30 minutes):
Positive in both cases
(XI) Glucose degradation product: L-lactic acid
[0021]

[0022]
The above-mentioned bacteriological properties (I) to (XI) are common to all of
the 5 bacterial strains and the Lactococcus lactis subsp. lactis-type strain ATCC 19435.
The growth temperature (XII), the salt-resistance (XIII), the pH-resistance (XIV), the
methylene blue-resistance (XV), the producibility of ammonia from arginine (XVI), and
the sugar fermentability (XVII) of each strain are shown in Table 1. The sugar
fermentation was examined with respect to 28 kinds of sugar using a medium for sugar
fermentation disclosed by Mitsuoka (Tomotari Mitsuoka, "The bacteriology of lactic acid
bacteria", Clinical Examination 18, Pages 1163 to 1172, 1974).
[0023]
It is apparent from the above-mentioned results that all of the 5 bacterial strains
have the bacteriological properties common to Lactococcus lactis subsp. lactis bacterial
strains. Thus, the 5 bacterial strains have been recognized to be bacterial strains
belonging to Lactococcus lactis subsp. lactis. On the other hand, it is apparent from the
above-mentioned bacteriological properties (XII) to (XVII) that the 5 bacterial strains are
different from the Lactococcus lactis subsp. lactis-type strain in that the 5 bacterial
strains do not have any abilities to ferment xylose.
[0024]
The 5 bacterial strains were deposited by the applicant at the International Patent
Organism Depositary, National Institute of Advanced Industrial Science and Technology,
(Central 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, JAPAN (postal code number:
305-8566)) as novel bacterial strains. The accession number of the Lactococcus lactis
subsp. lactis MCC852 is FERM BP-10742, that of the Lactococcus lactis subsp. lactis
MCC857 is FERM BP-10757, that of the Lactococcus lactis subsp. lactis MCC859 is
FERM BP-10744, that of the Lactococcus lactis subsp. lactis MCC865 is FERM
BP-10745, and that of the Lactococcus lactis subsp. lactis MCC866 is FERM BP-10746.
The Lactococcus lactis subsp. lactis MCC852, 859, 865, and 866 were deposited on
December 1, 2006, and the Lactococcus lactis subsp. lactis MCC857 was deposited on
January 10,2007.

[0025]
3. Test with respect to fermentability on 10% (W/W) reconstituted skim milk
medium.
Each 3% (V/V) bacterial strain starter was inoculated into a 10% (W/W)
reconstituted skim milk medium sterilized at 95°C for 30 minutes, and then cultured at
25, 30, or 37°C for 16 hours. After the obtained culture medium was rapidly cooled, the
curdled state was observed, and the pH and the viable count of the contained lactic acid
bacteria were measured. The viable count was measured using commercially available
BCP plate count agar (manufactured by Eiken Chemical Co., LTD.) flat plates. The
measurement results are shown in Table 2.
Lactococcus lactis subsp. lactis-type strain ATCC 19435 disclosed in Patent
Document 2 was used as a control strain.
[0026]

When each bacterial strain of Lactococcus lactis subsp. lactis MCC852, 857,
859, 865, and 866, that is, the bacteria of the genus Lactococcus to be used in the present
invention, was used, the pH of the culture medium was decreased to between 4.4 and 4.6
under any temperature conditions, and the culture medium was curdled. In addition, the
viable count of the contained lactic acid bacteria was approximately 1×109 CFU/g, and
thus the favorable proliferation and fermentability conditions were recognized.
On the other hand, when the Lactococcus lactis subsp. lactis-type strain ATCC

19435 was used, the pH of the culture medium was 5.5 or more and the culture medium
was not curdled under any temperature conditions. In addition, the viable count of the
lactic acid bacteria was significantly less at 30°C or higher, than that particularly of the
bacteria belonging to the genus Lactococcus according to the present invention.
[0028]
4. Co-cultivation test with Bifidobacterium longum
(1) Co-cultivation test with Bifidobacterium longum FERM BP-7787.
Lactococcus lactis subsp. lactis-type strain ATCC 19435 was used as a control
strain.
First, each culture of the 5 bacterial strains (Lactococcus lactis subsp. lactis
MCC852, 857, 859, 865, and 866) and Bifidobacterium longum FERM BP-7787 was
prepared in accordance with the method described in the following Example 1.
In addition, 1,000 mL of a 10% (W/W) reconstituted skim milk medium
containing 0.2% (W/W) yeast'extract (manufactured by Difco) was sterilized at 90°C for
30 minutes. Then, 30 mL of a culture of the Lactococcus lactis subsp. lactis-type strain
ATCC 19435 was inoculated into the reconstituted skim milk medium, and cultivated at
30°C for 16 hours to prepare a culture of the Lactococcus lactis subsp. lactis-type strain
ATCC 19435.
[0029]
1% (V/V) of each culture of the Lactococcus lactis subsp. lactis strains prepared
as above was inoculated with 1% (V/V) of the culture of the Bifidobacterium longum
FERM BP-7787 into a 10% (W/W) reconstituted skim milk medium sterilized at 90°C
for 10 minutes, and the mixture were cultivated at 37°C for 16 hours to obtain fermented
milk. The fermented milk was rapidly cooled, and the pH thereof and the viable count
of the contained Bifidobacterium were measured. Then, the resultant was further stored
at 10°C for two weeks, and the viable counts of the Bifidobacterium was measured at one
week and two weeks after initiation of storage. The viable count of the Bifidobacterium
was measured using TOS propionate agar (manufactured by YAKULT
PHARMACEUTICAL INDUSTRY CO., LTD.) flat plates. The measurement results

are shown in Table 3.
[0030]

[0031]
Each fermented milk prepared by using the Lactococcus lactis subsp. lactis
MCC852, 857, 859, 865, or 866 had a pH of approximately 4.5 and a Bifidobacterium
viable count of 5>
was stored at 10°C for two weeks, the survival rate of Bifidobacterium longum was 80%
or more.
On the other hand, fermentation of milk did not proceed with the Lactococcus
lactis subsp. lactis-type strain ATCC 19435, and the pH of the fermented milk was 5.0 or
more and storage thereof at 10°C was impossible. In addition, the Bifidobacterium
viable count immediately after the end of fermentation was approximately 1×108 CFU/g,
which was significantly small in comparison with the case where the bacteria belonging
to the genus Lactococcus according to the present invention were used.
[0032]
Thus, it is apparent that the 5 bacterial strains (the Lactococcus lactis subsp.
lactis MCC852, 857, 859, 865, and 866) are superior to other known bacterial strains of
the Lactococcus lactis subsp. lactis in terms of the properties promoting the growth of

Bifidobacterium longum FERM BP-7787 and the properties improving the survivability
thereof during storage.
It is also apparent that in the case where Bifidobacterium longum is co-cultured
with the Lactococcus lactis subsp. lactis that forms neither diacetyl nor acetoin as
described in Patent Document 2 is unlike the case where Bifidobacterium breve is used,
and neither the Bifidobacterium-proliferation promoting effects nor
Bifidobacterium-survivability improving effects disclosed in Patent Document 2 are
exhibited.
[0033]
(2) Co-cultivation test with Bifidobacterium longum-type strain ATCC 15707
The Bifidobacterium longum growth-promoting properties of the bacteria
belonging to the genus Lactococcus to be used in the present invention and
Bifidobacterium longum survivability-improving properties thereof during storage were
checked using the Bifidobacterium longum FERM BP-7787 and the Bifidobacterium
longum-type strain ATCC 15707.
First, a culture of the Lactococcus lactis subsp. lactis MCC857 and a culture of
the Bifidobacterium longum FERM BP-7787 were prepared in accordance with the
method described in the following Example 1.
In addition, a mixed culture of Streptococcus thermophilus and Lactobacillus
bulgaricus was prepared in accordance with the method described in the following
Example 2.
In addition, an 11% (W/W) skim milk medium containing 0.2% (W/W) yeast
extract was sterilized at 90°C for 30 minutes. Then, 10% (V/V) of the Bifidobacterium
longum-type strain ATCC 15707 was inoculated as a starter into the skim milk medium,
and cultivated at 37°C until the pH reached 4.6 to prepare a culture of the
Bifidobacterium longum-type strain ATCC 15707.
[0034]
1% (V/V) of the culture of the Lactococcus lactis subsp. lactis MCC857
prepared as described above, either 1% (V/V) of the culture of the Bifidobacterium

longum FERM BP-7787 or 1% (V/V) of the culture of the Bifidobacterium longum-type
strain ATCC 15707, and 0.01% (V/V) of the mixed culture of Streptococcus thermophilus
and Lactobacillus bulgaricus were inoculated into a 10% (W/W) reconstituted skim milk
medium sterilized at 90°C for 10 minutes, and cultivated at 37°C until the pH reached 4.6
to obtain fermented milk. After the obtained fermented milk was rapidly cooled, the
viable count of Bifidobacterium was measured. In addition, the fermented milk was
stored at 10°C for two weeks, and the viable count of Bifidobacterium longum was
measured at one week and two weeks after initiation of storage.
On the other hand, either 1.5% (V/V) of the culture of the Bifidobacterium
longum FERM BP-7787 prepared as described above or 1.5% (V/V) of the culture of the
Bifidobacterium longum-type strain ATCC 15707, and 0.4% (V/V) of the mixed culture
of Streptococcus thermophilus and Lactobacillus bulgaricus were inoculated into a 10%
(W/W) reconstituted skim milk medium sterilized at 90°C for 10 minutes, and cultivated
at 37°C until the pH reached 4.6 to obtain fermented milk as a control. The viable
Bifidobacterium count in the fermented milk was measured in the same manner. The
measurement results are shown in Table 4.
[0035]

[0036]
Both viable counts of the Bifidobacterium longum FERM BP-7787 and the
Bifidobacterium longum-type strain ATCC 15707 in fermented milk were significantly
increased by co-cultivating with the Lactococcus lactis subsp. lactis MCC857. In
addition, the survival rate of every Bifidobacterium stored at 10°C for two weeks was

30% or more: that of the Bifidobacterium longum FERM BP-7787 was 71% and that of
the Bifidobacterium longum-type strain ATCC 15707 was 31%.
In contrast, the survival rate of the Bifidobacterium longum FERM BP-7787
stored at 10°C for two weeks after cultivating in the absence of Lactococcus lactis subsp.
lactis MCC857 was 20% and no viable Bifidobacterium longum-type strain ATCC 15707
stored at 10°C for two weeks after cultivating in the absence of Lactococcus lactis subsp.
lactis MCC857 was detected.
The same results were obtained when every Lactococcus lactis subsp. lactis
MCC852, 859, 865, and 866 was used instead of the Lactococcus lactis subsp. lactis
MCC857.
[0037]
Thus, it is apparent that every Lactococcus lactis subsp. lactis MCC852, 857,
859, 865, and 866 also has excellent properties of both promoting the growth of
Bifidobacterium longum strains other than Bifidobacterium longum FERM BP-7787
having an excellent survivability during storage and improving the survivability of
Bifidobacterium longum strains other than Bifidobacterium longum FERM BP-7787
during storage.
[0038]
5. Comparative test with mixture of Lactococcus lactis subsp. lactis and
Lactococcus lactis subsp. cremoris disclosed in Patent Document 1.
The culture of Lactococcus lactis subsp. lactis MCC857, the culture of
Bifidobacterium longum-type strain ATCC 15707, and the mixed culture of Streptococcus
thermophilus and Lactobacillus bulgaricus were prepared in accordance with the method
described in the above 4(2).
[0039]
1% (V/V) of the culture of Lactococcus lactis subsp. lactis MCC857, 1% (V/V)
of the culture of Bifidobacterium longum-type strain ATCC 15707, and 0.01% (V/V) of
the mixed culture of Streptococcus thermophilus and Lactobacillus bulgaricus, prepared
in the above manner, were inoculated into a 10% (W/W) reconstituted skim milk medium

sterilized at 90°C for 10 minutes. The mixture was cultivated at 37°C until the pH
reached 4.6 to prepare fermented milk. The fermented milk was rapidly cooled and the
viable count of the contained Bifidobacterium was measured.
In contrast. 1% (V/V) of the culture of Bifidobacterium longum-type strain
ATCC 15707 prepared in the above-manner, and 2% (V/V) of a mixture "EZAL MA14"
composed of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris
(manufactured by Rhodia) were inoculated into a 10% (W/W) reconstituted skim milk
medium sterilized at 90°C for 10 minutes, as a control. The mixture was cultivated at
38°C until the pH reached 4.6 to prepare fermented milk. The viable count of
Bifidobacterium in the fermented milk was measured in the same manner. The mixture
"EZAL MAI4" corresponds to a mixture "EZAL MR014" (manufactured by Rhodia)
described in Patent Document 1.
[0040]
The viable Bifidobacterium count in fermented milk prepared using Lactococcus
lactis subsp. lactis MCC857 was 5.5×10 CFU/g. In contrast, no viable
Bifidobacterium cells were detected in a diluted solution obtained by diluting fermented
milk prepared using the mixture "EZAL MA14" by 106 fold, and thus the viable
Bifidobacterium count in the fermented milk was revealed to be 1 × 10 CFU/g or less.
[0041]
In other words, it was revealed that neither effects of promoting the growth of
Bifidobacterium nor such effects of shortening fermentation time, as mentioned in Patent
Document 1, were achieved when Bifidobacterium longum was co-cultivated with
Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris.
[0042]lAs described above, Lactococcus lactis subsp. lactis MCC852, 857, 859, 865,
and 866 (the bacteria belonging to the genus Lactococcus to be used in the present
invention) exhibit strong fermentability in the milk medium at a temperature suitable to
fermentation with Bifidobacterium, and also exhibit excellent effects of promoting the
growth of Bifidobacterium land improving the survivability thereof during storage, when

co-cultivated with Bifidobacterium longum. Thus, it is apparent that the bacteria have
properties which are not accompanied with conventionally known bacterial strains
belonging to the genus Lactococcus. In addition, it is expected that the bacteria to be
used in the present invention can produce fermented products with favorable taste, since
the bacteria produce neither diacetyl nor acetoin.
[0043]
Although a preculture medium used for cultivating Bifidobacterium and the
bacteria belonging to the genus Lactococcus in advance is not particularly limited
provided that the preculture medium is usually used, the preculture medium is preferably
a milk medium. The preculture medium is more preferably a reconstituted skim milk
medium, since the reconstituted skim milk medium can be easily handled. It is
preferable that the concentration of the reconstituted skim milk medium be 3% (W/W) or
more, and more preferably 8% (W/W) or more. In addition, the preculture medium may
contain growth-stimulating substances such as yeast extract or reducing agents such as
L-cysteine. It is particularly preferable that a growth-stimulating substance be
formulated in the preculture medium, since Bifidobacterium exhibits a low level of
proliferation in the milk medium. Specifically, a culture medium containing 0.1 to 1%
(W/W) of yeast extract may be used. The preculture medium is subjected to
sterilization for the use. The sterilization may be performed in accordance with a
conventional method, specifically performed by heating at 80 to 122°C for 5 to 40
minutes, preferably at 85 to 95°C for 5 to 35 minutes.
[0044]
The fermentation base medium to be used for fermentation with both
Bifidobacterium and bacteria belonging to the genus Lactococcus is not particularly
limited, provided that the fermentation base medium is usually used to produce
fermented milk. The fermentation base medium may be prepared, for example, by
formulating a sweetener such as sucrose, pectin, fruit, fruit juice, agar, gelatin, oil and fat,
flavor, coloring agent, stabilizer, reducing agent, or the like, in cow's milk, skim milk,
fresh cream, butter, whole milk powder, powdered skim milk, or the like, as needed,

followed by sterilizing, homogenizing, cooling, and the like, in accordance with
conventional methods.
[0045]
Although the inoculation ratio of Bifidobacterium to the bacteria belonging to
the genus Lactococcus, to be inoculated into the fermentation base medium as starters, is
not particularly limited, the inoculation ratio is preferably 100:1 to 1:10, and more
preferably 10:1 to 1:1. Although both amounts of Bifidobacterium and the bacteria
belonging to the genus Lactococcus to be inoculated in the fermentation base medium are
not particularly limited, it is preferable that the sum amount thereof be 0.01 to 10%
(V/V), more preferably 0.1 to 5% (V/V), with respect to the amount of the fermentation
base medium.
[0046]
The lactic acid bacteria to be used in the present invention may further contain
other lactic acid bacteria in addition to Bifidobacterium and the bacteria belonging to the
genus Lactococcus, unless the effects of the bacteria belonging to the genus Lactococcus
on the growth-stimulation of Bifidobacterium or the survivability-improvement thereof
during storage are disturbed. Although the other lactic acid bacteria are not particularly
limited, provided that the other lactic acid bacteria are usually used to produce fermented
milk, the other lactic acid bacteria are preferably Streptococcus thermophilus and
Lactobacillus bulgaricus. The inoculation ratio of the total content of Bifidobacterium
and the bacteria belonging to the genus Lactococcus to the total content of the other
lactic acid bacteria, to be inoculated as starters into a fermentation base medium, is not
particularly limited, unless the effects of the bacteria belonging to the genus Lactococcus
on Bifidobacterium are disturbed, it is preferable that the inoculation ratio be within a
range of 1,000:1 to 10:1.
[0047]
It is preferable that co-cultivation temperature in the method for producing
fermented milk according to the present invention be within a range of 30°C to 40°C,
more preferably 36°C to 38°C. Both Bifidobacterium and the bacteria belonging to the

genus Lactococcus to be used in the present invention can be sufficiently proliferate in
the above-mentioned temperature range. Although the cultivation time period is
suitably determined depending on the kind of fermented milk to be prepared, the
cultivation time period is preferably within a range of 5 to 18 hours.
[0048]
The fermented milk obtained by cultivation may be provided as a food product,
or may be homogeneously processed into a liquid state. In addition, fruit juice, fruit, or
the like, may be suitably formulated in the fermented milk. The fermented milk may be
put into a container by a conventionally-used method without any particular limitations.
[0049]
In the following, the present invention will be circumstantially explained by
indicating some examples. However, the present invention is not limited to the
following examples.
EXAMPLE 1
[0050]
1000 mL of 10% (W/W) reconstituted skim milk medium prepared by dissolving
10% by mass of skim milk powders (manufactured by Morinaga Milk Industry Co., Ltd.)
in water was sterilized at 90°C for 30 minutes, and then 30 mL of a seed culture of
Lactococcus lactis subsp. lactis MCC857 was inoculated thereinto, followed by
cultivating at 25°C for 16 hours. On the other hand, 1000 ml of an 11% (W/W) skim
milk medium containing 0.2% (W/W) yeast extract was sterilized at 90°C for 30 minutes,
and 100 mL of a seed culture of Bifidobacterium longum FERM BP-7787 was inoculated
thereinto, followed by cultivating at 37°C for 6 hours.
Apart from the above, 50L of a base medium prepared by mixing and dissolving
raw materials composed of skim milk powders, whole milk powders, pectin, and sucrose,
the base medium containing 0.5% (W/W) of butterfat, 8.0% (W/W) of nonfat milk solid
component, 5.0% (W/W) of sucrose, and 0.2% (W/W) of pectin, was sterilized at 90°C
for 10 minutes, followed by cooling at 40°C. 50 mL of the above-obtained culture of

the Lactococcus lactis subsp. lactis MCC857 precultured and 500 mL of the
above-obtained culture of Bifidobacterium longum FERM BP-7787 precultured were
inoculated into the sterilized base medium, followed by cultivating at 37°C for 16 hours
to obtain fermented milk. The fermented milk was immediately cooled while stirring,
and the cooled fermented milk was homogenized at a pressure of 15 MPa, followed by
putting the resultant into a glass container having a 200 mL capacity and then sealing the
container to obtain a yogurt drink. The obtained yogurt drink had a lactic acid content
of 0.68%, a pH of 4.64, and a viscosity of 75 mPa • s, and contained 7.0×108 CFU/g of
Bifidobacterium. When the yogurt drink was stored at 10°C for 21 days, the viable
count of Bifidobacterium was 5.2xl08 CFU/g, and the survival rate thereof was 74%.
EXAMPLE 2
[0051]
30 mL of a seed culture of Lactococcus lactis subsp. lactis MCC866 was
inoculated into 1000 mL of a 10% (W/W) reconstituted skim milk medium sterilized at
90°C for 30 minutes, and then cultivated at 25°C for 16 hours. On the other hand, 1000
mL of an 11% (W/W) skim milk medium containing 0.2% (W/W) yeast extract was
sterilized at 90°C for 30 minutes, and 100 ml of a seed culture of Bifidobacterium
longum FERM BP-7787 was inoculated thereinto, followed by cultivating at 37°C for 6
hours. Apart form the above, 50 mL of a mixed culture of Streptococcus thermophilus
(manufactured by HANSEN) and Lactobacillus bulgaricus (manufactured by HANSEN)
was inoculated into 1500 ml of a 10% (W/W) reconstituted skim milk medium sterilized
at 90°C for 30 minutes, and then cultivated at 37°C for 5 hours.
Apart from the above, 50 L of raw milk containing 3.0% (W/W) of burterfat and
9.0% (W/W) of nonfat milk solid component were heated at 70°C, homogenized at a
pressure of 15 MPa, sterilized at 90°C for 10 minutes, and then cooled at 40°C. Into the
thus sterilized base medium, 500 mL of the culture of Lactococcus lactis subsp. lactis
MCC866 precultured as described above, 500 mL of the culture of Bifidobacterium
longum FERM BP-7787, and 5 ml of the mixed culture of Streptococcus thermophilus

and Lactobacillus bulgaricus were inoculated. The resultant was put into a resin
container having a 500 mL capacity, and then the container was sealed. The bacteria
were cultivated at 37°C for 7 hours, and then immediately cooled. The thus obtained
fermented milk had a lactic acid content of 0.72%, and a pH of 4.55, and contained
5.8x10 CFU/g of Bifidobacterium. When the fermented milk was stored at 10°C for 21

days, the viable count of Bifidobacterium was 3.6×10 CFU/g, and the survival rate
thereof was 62%.
Example 3
[0052]
30 mL of a seed culture of Lactococcus lactis subsp. lactis MCC865 was
inoculated into 1000 mL of a 10% (W/W) reconstituted skim milk medium sterilized at
90°C for 30 minutes, and then cultivated at 25°C for 16 hours. On the other hand, 1000
mL of an 11% (W/W) skim milk medium containing 0.2% (W/W) yeast extract was
sterilized at 90°C for 30 minutes, and 100 ml of a seed culture of Bifidobacterium
longum FERM BP-7787 was inoculated thereinto, followed by cultivation at 37°C for 6
hours. Apart from the above, 50 mL of a mixed culture of Streptococcus thermophilus
(manufactured by HANSEN) and Lactobacillus bulgaricus (manufactured by HANSEN)
was inoculated into 1500 ml of a 10% (W/W) reconstituted skim milk medium sterilized
at 90°C for 30 minutes, and then cultivated at 42°C for 5 hours.
Apart from the above, 500 L of raw milk containing 3.4% (W/W) of butterfat
and 12.2%) (W/W) of nonfat milk solid component were heated at 70°C, homogenized at
a pressure of 15 MPa, sterilized at 90°C for 10 minutes, and then cooled at 40°C. Into
the thus sterilized base medium, 5 L of the culture of Lactococcus lactis subsp. lactis
MGC865 precultured as described above, 5 L of the culture of Bifidobacterium longum
FERM BP-7787, and 50 ml of the mixed culture of Streptococcus thermophilus and
Lactobacillus bulgaricus were inoculated, and then cultivated at 37°C for 7.5 hours,
followed by immediately cooling. The thus obtained fermented milk was further stirred
to pulverize curd, and the pulverized fermented milk was mixed with blueberry preserve

prepared by a conventional method at a ratio of 7:3.. The mixture was homogenized by
stirring, and then put into an oxidation barrier paper-container having a 120 mL capacity.
The thus obtained fermented milk with blueberry pulp had a lactic acid content of 0.80%,
and a pH of 4.45, and contained 5.5×10 CFU/g of Bifidobacterium. When the
fermented milk with blueberry pulp was stored at 10°C for 21 days, the viable count of
Bifidobacterium was 3.9×10 CFU/g, and the survival rate thereof was 71%.
INDUSTRIAL APPLICABILITY
[0053]
Since fermented milk containing a greater count of viable Bifidobacterium, than
ever before, even immediately before the end of use-by-date, can be produced in
accordance with the production method according to the present invention, the
production method is available in the field of production of fermented milk or the like.

WE CLAIM:
1. A method for producing a fermented milk, comprising:
performing fermentation using both bacteria belonging to a genus
Bifidobacterium and bacteria belonging to a genus Lactococcus as lactic acid bacteria,
wherein the bacteria belonging to the genus Lactococcus have bacteriological
properties of:
(1) a fermentability which curdles a 10% (WAV) reconstituted skim milk medium
when cultivated at a temperature of 25°C to 37°C for 16 hours;
(2) Bifidobacterium longum growth-promoting properties which lead a viable count
of Bifidobacterium longum of 5×108 CFU/g or more, when co-cultivated with

Bifidobacterium longum in the 10% (WAV) reconstituted skim milk medium until a pH
thereof is 4.4 to 4.6; and
(3) Bifidobacterium longum survivability-improving properties during storage,
which lead to a survival rate of Bifidobacterium longum of 30% or more, after
co-cultivation with Bifidobacterium longum in the 10% (WAV) reconstituted skim milk
medium until the pH thereof is 4.4 to 4.6, rapid cooling, and two weeks storage at 10°C.
2. The method for producing a fermented milk as claimed in claim 1, wherein the
bacteria belonging to the genus Lactococcus have no ability to ferment xylose and
produce neither diacetyl nor acetoin.
3. The method for producing a fermented milk as claimed in claim 1 or 2, wherein
the bacteria belonging to the genus Lactococcus are Lactococcus lactis subsp. lactis.
4. The method for producing a fermented milk as claimed in any one of claims 1 to
3, wherein the bacteria belonging to the genus Lactococcus comprise at least one
bacterial strain selected from the group consisting of Lactococcus lactis subsp. lactis
MCC852 (FERM BP-10742), Lactococcus lactis subsp. lactis MCC857 (FERM

BP-10757), Lactococcus lactis subsp. lactis MCC859 (FERM BP-10744), Lactococcus
lactis subsp. lactis MCC865 (FERM BP-10745), and Lactococcus lactis subsp. lactis
MCC866 (FERM BP-10746).
5. The method for producing a fermented milk as claimed in any one of claims 1 to
4, wherein the bacteria belonging to the genus Bifidobacterium are Bifidobacterium
longum.
6. The method for producing a fermented milk as claimed in claim 5, wherein a
bacterial strain of the Bifidobacterium longum is Bifidobacterium longum FERM
BP-7787.
7. The method for producing a fermented milk as claimed in any one of claims 1 to
6, wherein both Streptococcus thermophilus and Lactobacillus bulgaricus are further
used as the lactic acid bacteria.
8. A fermented milk prepared by the method for producing fermented milk as claimed in any
one of Claims 1 to 7.

ABSTRACT
The present invention relates to a method for producing a fermented milk,
including: performing fermentation using both bacteria belonging to the genus
Bifidobacterium and bacteria belonging to the genus Lactococcus as lactic acid bacteria,
wherein the bacteria belonging to the genus Lactococcus have the following
bacteriological properties: (1) fermentability which curdles a 10% (WAV) reconstituted
skim milk medium when cultivated at a temperature of 25°C to 37°C for 16 hours; (2)
Bifidobacterium longum growth-promoting properties which lead to a viable count of
Bifidobacterium longum of 5×108 CFU/g or more, when co-cultivated with
Bifidobacterium longum in the 10% (W/W) reconstituted skim milk medium until the pH
thereof is 4.4 to 4.6; and (3) Bifidobacterium longum survivability-improving properties
during storage, which lead to a survival rate of Bifidobacterium longum of 30% or more,
after co-cultivation with Bifidobacterium longum in the 10% (W/W) reconstituted skim
milk medium until the pH thereof is 4.4 to 4.6, rapid cooling, and two weeks storage at
10°C, and also relates to a fermented milk prepared by the production method.